Lac shuttle vectors

ABSTRACT

The invention discloses a Lac shuttle vector, comprising at least (a) a region which regulates a plasmid copy number; (b) an eukaryotic gene expression cassette, which comprises at least an eukaryotic gene transcriptional promoter sequence, a multiple cloning site and a transcriptional terminator sequence; (c) a lactic acid bacteria plasmid sequence, which comprises a plus origin of replication, and a nucleic acid sequence encoding for a protein which relates to the lactic acid bacteria plasmid replication; and (d) a non-antibiotic resistance selection gene and the promoter sequence thereof. The Lac shuttle vector features a non-antibiotic resistance gene as a selection marker, which is useful in pharmaceuticals and foods.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a Lac shuttle vector and uses thereof. More particularly, the Lac shuttle vector features a non-antibiotic resistance gene as a selection marker.

[0003] 2. Description of the Related Arts

[0004] Currently, most DNA vaccines are administrated by the uptake of bacteria such as Salmonella typhimurium or Listeria monocytegenes to mammals (Dietrich G., et al., 1998, Nature Biotech., 16:181-185; Lowrie, D. B., 1998, Nature Med., 4:147-148). The advantage is that the DNA vaccine can be directly incorporated into immune cells, or the immune system can be stimulated by the DNA vaccine, thereby enhancing the immune response. Usually, the effect produced by the administration of such bacteria is better than that produced by using DNA alone as a vaccine. However, such kinds of attenuated bacteria may be harmful to mammals being treated or may become pathogenic due to the mutagenesis of these bacteria.

[0005] During genetic engineering, plasmids have to bear a selection marker for selection of a cell (e.g. bacteria) containing the plasmid. Generally, most commercially available plasmids bear the antibiotic resistance gene such as an ampicillin resistance gene or a kanamycin resistance gene as a selection marker. Because most of these plasmids are used in laboratories, safety is not an important concern. However, some bacteria strains present in organisms may contain the antibiotic resistance genes via the natural transduction effect. If these plasmids are used as a pharmaceutical vaccine composition or food additive, organisms treated with the plasmids or the derivatives thereof may be jeopardized. In addition, the use of antibiotics can be problematic because of the potential for residual antibiotics in the purified DNA, which can cause an allergic reaction in a treated organism.

[0006] Therefore, the object of the present invention is to construct a vector without antibiotic resistance gene and use a harmless host cell to express heterologous genes in an organism or as a DNA vaccine or health food, thereby raising the safety and enhancing the immune response.

[0007] Conventional attenuated bacteria used for DNA vaccines may cause pathogenicity. Therefore, lactic acid bacteria are selected for a vaccine medium in the present invention to achieve the above object. Lactic acid bacteria are Gram-positive bacteria without pathogenicity, and are used in large quantity in the dairy and food industries. In addition, lactic acid bacteria are normal flora present in the digestive tract (Bomba A., et al., 1994, Vet. Med. (praha), 39:701-710; Nemcova R., et al., 1998, DTW Dtsch tierarztl wochenscher, 105:199-200). Further, certain chemical composition in the cell walls of lactic acid bacteria can stimulate the immune response in human (Vilma M. A., et al., 1996, Chem. Pharm. Bull., 44(12):2263-2267). If the lactic acid bacterial strains are modified so as to bear a heterologous gene or to be used as a DNA vaccine, the immunity or health of the organisms can be improved after uptake of such modified lactic acid bacteria. In addition, the recombinant plasmids or vectors can be prepared using genetic engineering to obtain more valuable lactic acid bacterial strains useful in the food industries.

SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention provides a Lac shuttle vector, comprising: (a) a region which regulates a plasmid copy number, wherein said region comprises an E. coli replication origin sequence; (b) an eukaryotic gene expression cassette, which comprises an eukaryotic gene transcriptional promoter sequence, a multiple cloning site and a transcriptional terminator sequence, wherein a heterologous gene is inserted into said multiple cloning site; (c) a lactic acid bacteria plasmid sequence, which comprises a plus origin of replication, and a nucleic acid sequence encoding for a protein which relates to the lactic acid bacteria plasmid replication; and (d) a non-antibiotic resistance selection gene and the promoter sequence thereof.

[0009] In another aspect, the present invention provides a kit for expression of a heterologous gene, wherein the Lac shuttle vector described above incorporating the heterologous gene is introduced into a suitable system to express the heterologous protein.

[0010] In still another aspect, the present invention provides a gene vaccine carrier, in which an antigenic gene derived from pathogens or cancers is incorporated into the Lac shuttle vector described above.

[0011] In yet another aspect, the present invention provides a method for selection of a host cell containing a vector, comprising: (i) introducing into said host cell the Lac shuttle vector described above, wherein the endogenous β-galactosidase gene of said host cell is not capable of producing a normal enzymatic function; and (ii) culturing said host cell transformed in step (i) under conditions which lactose is the only carbon source.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention will be more fully understood and further advantages will become apparent when reference is made to the following description of the invention and the accompanying drawings in which:

[0013]FIG. 1 is a diagram showing the constructs of the pCLP1 and pCLP2.

[0014]FIG. 2 is a diagram showing the construct of the pCRII/Em^(r)P-β-galactosidase.

[0015]FIG. 3 is a diagram showing the constructs of the pCLP3, pCLP4, pCLP5, and pCLP6.

[0016]FIG. 4 is a diagram showing the constructs of the pCLP7 and pCLP8.

[0017]FIG. 5(A)-(G) is a diagram showing the nucleotide sequence of the Lac shuttle vector pCLP7 (SEQ ID NO: 1).

[0018]FIG. 6(A)-(G) is a diagram showing the nucleotide sequence of the Lac shuttle vector pCLP8 (SEQ ID NO: 2).

[0019]FIG. 7 is a diagram showing the stability test of vectors pCLP7 and pCLP8.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The term “Lac shuttle vector” used herein refers to a vector in which both plasmid replication origins of E. coli and Lactobacillus are present, so that the vector can replicate and proliferate both in E. coli and lactic acid bacteria, thereby breaking down the barrier between bacteria species.

[0021] For the purpose described above and for convenient manipulation subsequently, the Lac shuttle vector of the present invention comprises the replication origin, Col E1, which is necessary for the vector replication in E. coli.

[0022] For the purpose of expressing a heterologous protein in an eukaryotic cell or being used as a DNA vaccine carrier, the Lac shuttle vector of the present invention comprises an eukaryotic gene expression cassette, which comprises at least an eukaryotic gene transcriptional promoter sequence, a multiple cloning site and a transcriptional terminator sequence, wherein a desired heterologous gene is inserted into the multiple cloning site in the vector. Those skilled in this art will be aware that the elements that can be used in the vector described above are not limited. For example, the eukaryotic gene transcriptional promoter sequence suitable for the present invention includes cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, Rous sarcoma virus (RSV) promoter, etc.

[0023] The term “vector”, as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids” which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.

[0024] As used herein, the term “regulate”, or variations such as “regulates” or “regulating”, will be understood to imply sequences involved in control of a response or action. This includes sequences involved in regulating, controlling or affecting the expression of genes, or the replication, selection or maintenance of a plasmid. Examples include attenuators, operators, and promoters.

[0025] The term “replication origin” as used herein refers to a nucleotide sequence at which DNA synthesis for the purpose of replicating the nucleic acid sequence begins. This is generally termed an ori site. Bacteria generally have a single ori site, whereas there are many ori sites on each eukaryotic chromosome. This term includes replicons, which as used herein refers to a genetic element that behaves as an autonomous unit during DNA replication. In bacteria, the chromosome functions as a single replicon, whereas eukaryotic chromosomes contain hundreds of replicons in series.

[0026] The term “antibiotic resistance” as used herein refers to the acquisition of tolerance to a specific antibiotic by a microorganism that was previously adversely affected by the drug. Such resistance generally results from a mutation or the acquisition of resistance due to plasmids containing the resistance gene transforming the microorganism.

[0027] In a preferred embodiment of the present invention, the eukaryotic gene transcriptional promoter sequence is cytomegalovirus promoter (hereinafter abbreviated as “pCMV”), and transcriptional terminator sequence is bovine growth hormone polyadenylate (BGH polyA) region, which terminate and stabilize the synthesis of messenger RNA (mRNA).

[0028] There are many natural plasmids contained in Lactobacillus strains, in which the replication origins and genes encoding necessary proteins are suitable for the vector construction of the present invention. It is known that there are three different kinds of natural plasmids contained in Lactobacillus plantarum (ATCC 8014, CCRC 10357) with 2.1, 10.5, and 38.8 kb in size, respectively (Yan T-R, et al., 1996, Chinese Agri. Chem. Soc., 34:723-731). Due to the requirements of convenience and stabilization, the smallest one of the three plasmids is selected for the vector of the present invention. The plasmid of 2.1 kb in size contains a plus origin of replication, an open reading frame, and a replication control region with a sequence group of 17 nucleotides repeated 13 times; wherein the open reading frame translates a protein (Rep A protein) of 317 amino acids in length, which possesses the function associated with plasmid replication (Bouia A., et al., 1989, Plasmid, 22:185-192; Bringel F., et al., 1989, Plasmid, 22:193-202). The present invention chooses a suitable restriction enzyme such as Bc1I to linearize the plasmid under the condition that two important elements described above are not destroyed, so as to facilitate the linear plasmid's incorporation into the vector of the present invention.

[0029] The β-galactosidase gene in the genome of Lactobacillus delbrueckii (subsp. bulgaricus) is selected as a selection marker gene in lieu of antibiotic resistance gene. The product of β-galactosidase gene is a metabolic enzyme, which can hydrolyze lactose to glucose and galactose. (Schmidt B. F., et al., 1989, J. Bacteriol., 171:625-635). Therefore, when bacteria lack the enzyme (e.g., E. coli strain JM109) and grow under conditions which lactose is the only carbon source, the bacteria have to utilize the β-galactosidase encoded by the gene present in the plasmid to metabolize lactose to obtain the glucose required for growth (Hashiba H., 1992, Biosci. Biotech. Biochem., 56:190-194).

[0030] The β-galactosidase gene selected for the vector of the present invention is expressed under the control of a promoter. Preferably, the promoter is a strong transcriptional promoter to overexpress the gene product (Hashiba H., 1992, Biosci. Biotech. Biochem., 56:190-194). In one preferred embodiment of the present invention, the promoter is erythromycin resistance gene promoter (hereinafter abbreviated as “Em^(r)P”).

[0031] The advantages of using β-galactosidase gene as a selection marker include not only replacing antibiotic resistance gene, but also meeting the safety requirements of pharmaceuticals and foods.

[0032] In accordance with the construction of the present invention, the function of the β-galactosidase encoded by the gene of the host cell itself (i.e., endogenous β-galactosidase) has to be destroyed (i.e., the host cell can not express β-galactosidase with normal enzyme activity) so that host cell must rely on the recombinant vector of the present invention for survival. The present invention uses N-methyl-N′-nitro-N-nitrosoguanidine; MNNG) to treat the host bacteria in the log phase. MNNG is an alkylating agent, which facilitates acting on guanine and thymine to cause DNA mutation. Therefore, a bacterial strain with a defective β-galactosidase gene can be selected.

[0033] The host bacterial strain suitable for use in the present invention includes, but is not limited to Lactobacillus casei (subsp. casei) . The advantages of using this strain include that: (1) this bacterial strain is one of the strains used for the production of cheese that meets the safety requirement of foods; (2) this bacterial strain can be anchored and colonized in the intestine; and (3) the shuttle vector of the present invention comprises a plus origin of replication of lactic acid bacterial plasmid, so that the vector can replicate and segregate stably in lactic acid bacterial host (Leer R., et al., 1992, Mol. Gen. Genet., 234:265-274; Posno M., et al., 1991, Appl. Environmental Microbiol. 57:1822-1828). It is to be understood that lactic acid bacterial strain is one of the Gram-positive bacteria, and the shuttle vector of the present invention bear both replication origins of E. coli and Lactobacillus. Thus, those skilled in this art will be aware that any Gram-positive bacterium with similar genetic properties to lactic acid bacteria can also be used as host cells in the present invention. That is, any appropriate bacterial strain that is mutated to possess defective β-galactosidase gene, or any appropriate bacterial strain whose endogenous β-galactosidase gene does not have normal enzyme activity, can be used in the present invention. In a preferred embodiment, Lactobacillus casei (subsp. casei) is treated with MNNG, followed by screening with X-gal and a selection medium to obtain a Lac mutant designated Ana-1. Ana-1 is deposited with American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, Va. 20110, USA, on Nov. 10, 2000 and assigned ATCC Accession No. PTA-2662.

[0034] In the multiple cloning site of the Lac shuttle vector of the present invention, suitable antigenic genes derived from pathogens or cancers can be inserted to obtain a gene vaccine carrier. This gene vaccine carrier is then transformed into a bacterial strain with defective a β-galactosidase gene, followed by administration to organisms orally or by injection (for example, intravenously, intraarterially, subcutaneously, intraperitoneally, intracranially, or intramuscularly). Thus, the gene vaccine carrier can be incorporated into cells of organisms by endocytosis or phagocytosis. In addition, due to the construction of an eukaryotic gene transcriptional promoter sequence (e.g. pCMV), the antigenic genes in the multiple cloning site can be expressed immediately.

[0035] The use of the Lac shuttle vector in combination with the host cell with defective β-galactosidase gene as a gene vaccine has advantages as follows: (1) lactic acid bacteria is neither toxic nor pathogenic; (2) certain chemical compositions in the cell wall of lactic acid bacteria can stimulate and enhance the immune response in mammals; (3) the dosage of Lac shuttle vector used as a gene vaccine is much lower than that of the direct injection of a DNA vaccine; the former is about less than 1 mg/dose, whereas the later is about 100 mg/dose and the injection site is limited to, for example, the site near the body surface; and (4) the safety of Lac shuttle vector is much higher than the use of a viral vector.

[0036] In the multiple cloning site of the Lac shuttle vector of the present invention, a heterologous gene can also be inserted to obtain a recombinant expression vector. The heterologous protein of interested thus can be in vivo or in vitro overexpressed in a suitable eukaryotic cell by the eukaryotic gene transcriptional promoter in the vector. Those skilled in this art will be aware that the eukaryotic gene transcriptional promoter can be replaced with a suitable prokaryotic gene transcriptional promoter, so that the heterologous protein of interested can be overexpressed in an appreciate prokaryotic cell.

[0037] The transformation of vectors into a cell can be achieved via various mechanisms known to those skilled in this art. For example, transformation (including treatment with divalent cation, DMSO, reducing reagent, hexamminecobalt chloride and so on), electroporation or particle bombardment.

[0038] Without intending to limit it in any manner, the present invention will be further illustrated by the following examples.

EXAMPLE

[0039] The bacterial strains described above and used in the following examples included Lactobacillus delbrueckii (subsp. bulgaricus) (CCRC 14008), Lactobacillus plantarum (CCRC 10357), and Lactobacillus casei (subsp. casei) (CCRC 10697), which are available from Culture Collection and Research Center (CCRC), Hsinchu, Taiwan. All three bacteria were grown in Lactobacilli MRS broth (including 10 g/L proteose peptone No.3, 10 g/L beef extract, 5 g/L yeast extract, 20 g/L dextrose, 1 g/L Tween-80, 2 g/L ammonium citrate, 5 g/L CH₃COONa, 0.1 g/L MgSO₄·7H₂O, 0.05 g/L MnSO₄·H₂O, 2 g/L K₂HPO₄, pH 6.2-6.5) at 37° C.

Example 1 Preparation of Lac⁻ Mutant Host Strain (Ana-1)

[0040] 20 μl overnight culture of Lactobacillus casei (subsp. casei) was inoculated to 1 ml MRS broth and incubated at 37° C. for 4 hours. A pellet was obtained by spin-down and then washed twice with PBS buffer (50 mM potassium phosphate, 150 mM NaC1, pH 7.2). The bacterial pellet was resuspended in 0.9 ml PBS buffer and treated with 0.1 ml MNNG stock (N-methyl-N′-nitro-N-nitrosoguanidine; 5 mg/ml in 0.05 M acetic acid). After slow rotation at 37° C. for 1 hour, bacteria were pelleted down and washed three times with PBS buffer, and then resuspended in 0.1 ml MRS broth. Ten-fold serial dilution was performed with MRS broth. 0.1 ml bacteria in MRS from each dilution was spread to a diameter 100 mm agar plate, which has been spread with 40 μl of X-gal (20 mg/ml). These plates were incubated at 37° C. for 1-3 days. White colonies were selected as Lactobacillus casei (subsp. casei) Lac⁻ mutant (Ana-1).

Example 2

[0041] 1. Isolation of Lactobacillus plantarum Plasmid

[0042]Lactobacillus plantarum were collected from 15 ml MRS/overnight cultured broth. The bacteria were lysed with 4.755 ml solution I (6.7% sucrose, 50 mM Tris-HCl pH 7.6, 1 mM EDTA pH 8.0, and 100 μg/ml lysozyme) at 37° C. for 20 min. After addition of 482 μl solution II (50 mM Tris-HCl pH 7.6 and 0.25 M EDTA) and 276 μl solution III (20% SDS, 50 mM Tris-HCl pH 7.6, and 20 mM EDTA), the mixture was incubated at 37° C. for 20 min and vigorously shaken for 30 sec, followed with addition of 1.276 ml of 3 N NaOH and rotation for 10 min, and addition of 496 μl of 2 M Tris and rotation for another 10 min. For extraction of bacterial protein, bacterial lysate were added with 717 μl of 5 M NaCl and 700 μl phenol saturated with 3% NaCl. After centrifugation, the aqueous phase was extracted with equal volume of chloroform-isoamylalcohol (24:1). After mixing and centrifugation, the aqueous phase was precipitated with equal volume of isopropanol at 0° C. for 1 hour and then centrifuged for 5 min to obtain DNA pellet. The DNA pellet was air dried and dissolved in 20 μl H₂O. The quality and quantity of the plasmid DNA was estimated by 1% agarose electrophoresis and ethidium bromide stain.

[0043] 2. Cloning of Lactobacillus Plantarum 2.1 kb Plasmid

[0044] The plasmids isolated from Lactobacillus plantarum were fractionated by electrophoresis on 1% agarose gel and purified by GENECLEAN III kit (Bio 101, La Jolla, Calif.). To generate plasmids pCLP1 and pCLP2, the 2.1 kb plasmid was digested with restriction enzyme BclI and ligated into the BglII site of pCLP0, which consists of CMV promoter, BGH polyA sequence, ColE1 replication origin, and Amp^(R) open reading frame (see FIG. 1).

Example 3

[0045] 1. Isolation of Chromosomal DNA from Lactobacillus Delbrueckii (Subsp. bulgaricus)

[0046]Lactobacillus delbrueckii (subsp. bulgaricus) were collected from 20 ml MRS/overnight cultured broth. The bacterial pellet was resuspended in 1 ml TES buffer (100 mM Tris, 20 mM EDTA, 20% sucrose, and 1 mg/ml lysozyme) at 37° C. for 30 min. The cells were then subjected to five freeze-thaw cycles by freezing in a dry ice-ethanol bath and thawing in a 37° C. water bath. The cells were lysed by addition of ½ volume of 1% SDS (sodium dodecyl sulfate) solution. The chromosomal DNA was purified by triple phenol extractions. The DNA was finally precipitated with ethanol, air dried, and then dissolved in H₂O.

[0047] 2. Cloning of β-galactosidase Gene

[0048] The β-galactosidase gene was amplified from chromosomal DNA of Lactobacillus delbrueckii (subsp. bulgaricus) by polymerase chain reaction (PCR). The PCR amplification consisted of 0.075 units Pfu Turbo™ DNA polymerase (STRATAGENE®, La Jolla, Calif.), 1 μM each of forward (5′-aagctcatgaTTGGCAGCCAGTCTCCGGGC-3′) and reverse primers (5′-gacctcatgaACCGTCGCTAGCGACACGCC-3′). PCR was carried out in 4 stages: (i) 95° C. for 5 min; (ii) 94° C. for 30 sec, 54° C. for 30 sec, 72° C. for 3 min, ×30 cycles; (iii) 72° C. for 10 min; and (iv) hold at 4° C. The amplified DNA product was estimated by 0.8% agarose electrophoresis and ethidium bromide stain, followed with purification by GENECLEAN III kit (Bio 101, La Jolla, Calif.). The purified 3 kb β-galactosidase DNA fragment was ligated into EcoRV site of pcDNA3 vector (INVITROGENE). The ligation mixture was transformed into E. coli strain DH5α. The blue-color clones containing the plasmid bearing β-galactosidase gene were selected from X-gal/Amp LB agar plate.

Example 4

[0049] Cloning of erythromycin^(r) Enh/promoter (Em^(r)P) DNA Fragment and Em^(r)P-β-galactosidase DNA Fragment

[0050] The plasmid pVA838 obtained from CCRC (Hsinchu, Taiwan) was used as template for cloning of (Em^(r)P) DNA fragment via PCR. The PCR amplification consisted of 0.075 units Pfu Turbo™ DNA polymerase (STRATAGENE®), 1 μM each of forward (5′-TTAACGATCGTTAGAAGCAAACTTAAGAGTG-3′) and reverse primers (5′-TTAACGATCGATGTAATCACTCCTTCT-3′). PCR was carried out in 4 stages: (i) 95° C. for 5 min; (ii) 94° C. for 30 sec. 50° C. for 30 sec, 72° C. for 30 sec, ×30 cycles; (iii) 72° C. for 10 min; and (iv) hold at 4° C. The amplified DNA product was estimated by 1% agarose electrophoresis and ethidium bromide stain, followed with purification by phenol/chloroform extraction and ethanol precipitation. The purified 120 bp Em^(r)P DNA fragment was ligated into the pCRII vector (INVITROGENE). These clones bearing the pCRII/Em^(r)P plasmid were selected from X-gal/Amp LB agar plate as white colonies and further checked by PCR and restriction enzyme analysis.

[0051] The pCRII/Em^(r)P plasmid was purified by GFX Micro plasmid Prep Kit (Amersham Pharmacia Biotech.), then linearized with BamHI and blunted at the end with T4 DNA polymerase. For construction of plasmid pCRII/Em^(r)P-β-galactosidase, the β-galactosidase DNA fragment was amplified from pcDNA3/β-galactosidase plasmid by PCR and ligated to the blunt end of linearized pCRII/Em^(r)P. These clones bearing the pCRII/EM^(r)P-β-galactosidase plasmid were selected from X-gal/Amp LB agar plate as blue colonies and further checked by PCR and restriction enzyme analysis (see FIG. 2).

Example 5 Cloning of Lac Shuttle Vector

[0052] After phosphorylation of the 5′-end of Em^(r)P-β-galactosidase DNA fragment amplified by PCR, the DNA fragment was purified by GENECLEAN III kit (Bio 101, La Jolla, Calif.) and ligated to the NruI site of plasmids pCLP1 and pCLP2 to obtain plasmids pCLP3-6 (see FIG. 3). The ligation mixture was electroporated to E. coli JM109 strain. These clones bearing the plasmid containing Em^(r)P-β-galactosidase gene were selected from X-gal/Amp LB agar plate as blue colonies and further checked by PCR and restriction enzyme analysis.

[0053] To delete the ampicillin resistance gene from the shuttle vectors pCLP3 and pCLP5, these two plasmids were digested with BspHI. The 1 kb DNA fragment containing the ampicillin resistance gene was discarded by gel-elution of the larger DNA fragment. The purified DNA fragments were further ligated and transformed into E. coli JM109 strain. These clones bearing the plasmid were selected by growth on L-M9 plate (including 6 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 5 g/L NaC1, 1 g/L NH₄Cl, 2 mM MgSO₄, 0.1% lactose, 0.1 mM CaCl₂, 2 mM proline, and 50 μM thiamine) but not on Amp/LB agar plate, and further checked by PCR and restriction enzyme analysis (see FIG. 4).

Example 6 Preparation of Ana-1 Competent Cell

[0054] To prepare the competent cell for electroporation, 1 ml overnight culture of Lac⁻ mutant host strain from Lactobacillus casei (subsp. casei) (Example 1) was inoculated to 50 ml MRS broth supplied with 1.25% glycine and incubated at 37° C. for 3 hours. The cells were pelleted down, washed four times with ice-cold poration/storage buffer (0.5 M sucrose and 10% glycerol) and resuspended in 0.5 ml ice-cold poration/storage buffer. The plasmid DNA was purified by QIAprep Miniprer Kit (QIAGEN). 1 μg plasmid DNA was mixed with 100 μl competent cell in a disposable cuvette (STRATAGENE®; 0.2 cm interelectrode distance). A single pulse of 2500 volts (600 ohm, 25 μF) was delivered to this DNA-cell mixture. Following the pulse, the cell suspension was directly diluted with 0.4 ml MRS broth and incubated at 37° C. for 1.5 hours to allow expression of the β-galactosidase gene. The transformants were selected by plating 100 μl of the dilution of cell suspension on L-MRS agar plate, which the formula was the same as MRS, except that the dextrose was replaced with 2% lactose.

[0055] Refer to FIG. 1. Because the sequence of 5′-sticky end formed by the digestion of BclI and BglII are identical, the linear lactic acid bacteria plasmid (LP) is inserted into PCLP0 in two different directions. The resulting different plasmids are obtained by PCR amplification and restriction enzymes analysis, in which the plasmid with same direction of Rep A gene and CMV promoter is designated pCLP1, whereas the plasmid with opposite direction of Rep A gene and CMV promoter is designated pCLP2. Both pCLP1 and pCLP2 can replicate in E. coli for many generations and their restriction mappings can remain, indicating the copy numbers and stability of the recombinant shuttle vectors in E. coli are not affected after the lactic acid bacteria plasmid is inserted into the E. coli plasmid (pCLP0).

[0056] When the plasmids bearing β-galactosidase gene is transformed into bacteria with a defective β-galactosidase gene in the chromosome, for the purpose of survival and replication on the selective medium (with lactose but not glucose as the only carbon source), the bacteria have to overexpress the products of the selection marker gene such as β-galactosidase to metabolize the required elements. Therefore, the strong transcriptional promoter Em^(r)P is used in the shuttle vector of the present invention. Referring to FIG. 3, after construction of Em^(r)P-β-galactosidase, the DNA fragment is inserted into plasmids pCLP1 and pCLP2. Due to the blunt end ligation, the EM^(r)P-β-galactosidase DNA fragment is inserted in two different directions. Four different plasmids are obtained by PCR amplification and restriction enzymes analysis, and designated pCLP3, pCLP4, pCLP5, and pCLP6, respectively. The subculture of these plasmids is then carried out for many generation, wherein the copy numbers of pCLP4 and pCLP6, and the growth of host cells transformed by these two plasmids are significantly decreased. It is thus inferred the plasmids pCLP4 and pCLP6 are structurally unstable, whereas pCLP3 and pCLP5 are relatively stable.

[0057] To construct the plasmid having non-antibiotic resistance gene as the selection marker, the ampicillin resistance genes of pCLP3 and pCLP5 are then deleted to obtain the plasmids pCLP7 and pCLP8 (Referring to FIG. 4). After transforming into E. coli, the bacteria can grow well in the medium which the lactose is the only carbon source. In addition, the restriction mapping analysis shows the structures of pCLP7 and pCLP8 are stable after subculture. Therefore, the stability of the shuttle vectors pCLP7 and pCLP8 makes them useful during genetic engineering. The nucleotide sequence of pCLP7 is shown in FIG. 5 (SEQ ID NO: 1) and that of pCLP8 is shown in FIG. 6 (SEQ ID NO: 2). Further, the restriction mapping of original vectors pCLP7 and pCLP8 is shown in FIG. 7A, in which lane 1 is a DNA marker (Gene Ruler™ 1 kb Ladder, MBI), lanes 2, 3 are untreated vectors, lanes 3, 7 are vectors treated with XbaI, lanes 4, 8 with EcoRI, lanes 5, 9 with HindIII, and lane 10 is another DNA marker (Gene Ruler™ 100 bp Ladder, MBI), wherein lanes 2-5 are vectors pCLP7 and lanes 6-9 are vectors pCLP8.

[0058] When the Lac shuttle vectors pCLP7 and pCLP8 of the present invention are transformed into Lac⁻ mutant strain Ana-1 (with defective β-galactosidase gene), the bacterial strain grows well in the selective medium. Moreover, the restriction mapping analysis also shows the structures of pCLP7 and pCLP8 are stable after 100 generations of subculture. Referring to FIG. 7B, it shows the mapping of vectors after 100 generations of subculture, in which lane 1 is a DNA marker (Gene Ruler™ 100 bp Ladder, MBI), lane 2 is another DNA marker (Gene RulerT™ 1 kb Ladder, MBI), lanes 3, 7 are untreated vectors, lanes 4, 8 are vectors treated with XbaI, lanes 5, 9 with EcoRI, and lanes 6, 10 with HindIII, wherein lanes 3-6 are vectors pCLP7 and lanes 7-10 are vectors pCLP8.

[0059] While the invention has been particularly shown and described with the reference to the preferred embodiment thereof, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the spirit and scope of the invention.

1 6 1 8115 DNA Lactobacillus plantarum 1 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca 60 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta 120 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat 180 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga ctatttacgg 240 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac 300 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt 360 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg 420 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc 480 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt 540 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 600 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg 660 actcactata gggagaccca agcttggtac cgagctcgga tccactagta acggccgcca 720 gtgtgctgga attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag 780 ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct 840 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 900 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 960 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac 1020 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc 1080 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 1140 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 1200 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 1260 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 1320 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 1380 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 1440 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 1500 cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 1560 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 1620 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 1680 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 1740 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 1800 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 1860 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 1920 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 1980 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2040 cccgaaaagt gccacctgac gtcgacggat cgggagatca acggtaaatc cgttggcata 2100 tccctttttt gttgtcagct tgctgacttc tgatacaggt tttagcatta ctccaattta 2160 tttggagtgt aagtgcacat tatcatgtag tgcgcattat catgtagtgc gcattatcat 2220 gtagtgcgca ttatcatgta gtgcgcatta tcatgtagtg cgcattatca tgtagtgcgc 2280 attatcatgt agtgcgcaca ttatcatgta cattatcatg tagtgcgcat tatcatgtag 2340 tgcgcacatt atcatgtagt gcgcattatc atgtagtgcg cattatcatg tagtgcgcac 2400 ttacacacaa catgaagttg tgttgtgcta aacccatcaa aacctgcatc agatttcgcg 2460 ttgctcaaac gtaactgact tgcgtcagtt tggaacattc aaaaataaat aagttcagtc 2520 gctagctcct tcgaactttt ttatttttga acgttaattt taaaggctct tatttgcgtt 2580 ctaagcgatt ttagctaaca gttagctatc taactgtctg tcaacggtaa atcgacttag 2640 aggggcttat tgagccttac aggcgatatt agcccctctt ggaggcttta aggagttgat 2700 agactagaca ataccaaaag cctgacgtct tggaaaacaa gcccttgttt tcccgagccc 2760 agcggcggca agcgttacgg tccagctggt tcagctggtc agtgtggctg aaagccacgg 2820 tttaaaaaaa gcagttcagc ggtttttgct gatctgcttt ttggggttta aaaacgcaat 2880 ttttggcgtt ttcttcttat cttgatacta ttagcaacaa ctagtttttt aaaatcaagc 2940 ttgattaggc ttaattgggc ttgtatccat tgattttata ggcttttggt gtattattag 3000 ggttataaat tggttgaaag aaagacaaaa taaaaaccca cgtgcaaatt cctagtttgg 3060 ccgctcggaa cacgtgagtt gattatcatt tgcgatttat agcctattct aggggaaaag 3120 ccctatgatg tcaaggttat aagcttattg aaaaagatag tcagctcctt cacgtggata 3180 aactggagga gctttttatg tcagaaattt ttgaagataa aactgaaaat ggcaaagtta 3240 gaccttggcg agaacggaag attgaaaatg tgcgctatgc cgaatatttg gcaatcttag 3300 aatttaaacg ggcacatgat gtacggggtt gtggtgaagt tttgcgtttt cgtaagattg 3360 gcgagcactt aaaactttat caaacgtggt tttgtcataa acgattgtgt ccattgtgta 3420 attggagaag gagcatgaaa aactcgagcc agttaaaaca aattattgcg gaagcagttg 3480 caagagagcc taaaggacgg tttttgtttt taactttaac cgttaaaaac gctcattcag 3540 cagaggagtt aaaagtgtct ttaagagctt tgactaaagc ctttaataag ctaactcgct 3600 ataaaaaagt gactaaaaat ttattgggtt atttacgttc aacggaaatt accgttaatg 3660 aacaagacgg gtcatataat caacacttgc atgtgttgct gtttgtaaaa tcaagttatt 3720 ttaagaattc aaataattat ttagcacaag cagaatgggc aaaattatgg caaaaagcct 3780 tgaaagttga ttatgagcct gtggtgcatg tgcaggctgt taaagctaac aaacgtaaag 3840 gaactgactc tttgcaagct agtgccgaag aaacggcgaa atacgaggta aaatcagctg 3900 attatatgac ggctgatgat gagcgtaatt tggtggtgat taaaaatttg gagtatgcct 3960 tagctggaac acgacaaatc agctatggtg gattattaaa gcaaattaag caagatttga 4020 aacttgaaga tgttgagaat ggtgatttag ttcatgttgg cgatgaagat tacaccaaag 4080 agcaaatgga agctgcggaa gaagttgtcg caaaatggga ttttaataaa caaaattatt 4140 ttatttggta aagagaatgt caggatatga tctcccgatc ccctatggtc gactctcagt 4200 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag 4260 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat 4320 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgttagaa gcaaactaag 4380 agtgtgttga gtagtgcagt atcttaaaat tttgtataat aggaattgaa gttaaattag 4440 atgctaaaaa tttgtaatta agaaggagtg attacatgat tggcagccag tctccgggca 4500 attaatgaac ttggacatgg ttgacgaccc ggtctttgca agccgaattc gaccacactg 4560 gcggccgtta ctagggtatc gatccgataa aaagttaggc gacggctttg ccctggtgcc 4620 agcagacggt aaggtctacg cgccatttgc cggtactgtc cgccagctgg ccaagacccg 4680 gcactcgatc gtcctggaaa atgaacatgg ggtcttggtc ttgattcacc ttggcctggg 4740 cacggtcaaa ttaaacggga ctggctttgt cagctatgtt gaagagggca gccaggtaga 4800 agccggccag cagatcctgg aattctggga cccggcgatc aagcaggcca agctggacga 4860 cacggtaatc gtgaccgtca tcaacagcga aactttcaca aatagccaga tgctcttgcc 4920 gatcggccac agcgtccaag ccctggatga tgtattcaag ttagaaggga agaattagaa 4980 aatgagcaat aagttagtaa aagaaaaaag agttgaccag gcagacctgg cctggctgac 5040 tgacccggaa gtttacgaag tcaatacaat tcccccgcac tccgaccatg agtccttcca 5100 aagccaggaa gaactggagg agggcaagtc cagtttagtg cagtccctgg acggggactg 5160 gctgattgac tacgctgaaa acggccaggg accagtcaac ttctatgcag aagactttga 5220 cgatagcaat tttaagtcag tcaaagtacc cggcaacctg gaactgcaag gctttggcca 5280 gccccagtat gtcaacgtcc aatatccatg ggacggcagt gaggagattt tcccgcccca 5340 aattccaagc aaaaatccgc tcgcttctta tgtcagatac tttgacctgg atgaagcttt 5400 ctgggacaag gaagtcagct tgaagtttga cggggcggca acagccatct atgtctggct 5460 gaacggccac ttcgtcggct acggggaaga ctcctttacc ccaagcgagt ttatggttac 5520 caagttcctc aagaaagaaa ataaccgcct ggcagtggct ctctacaagt attcttccgc 5580 ctcctggctg gaagaccagg acttctggcg catgtctggt ttgttcagat cagtgactct 5640 tcaggccaag ccgcgtctgc acttggagga ccttaagctt acggccagct tgaccgataa 5700 ctaccaaaaa ggaaagctgg aagtcgaagc caatattgcc taccgcttgc caaatgccag 5760 ctttaagctg gaagtgcggg atagtgaagg tgacttggtt gctgaaaagc tgggcccaat 5820 cagaagcgag cagctggaat tcactctggc tgatttgcca gtagctgcct ggagcgcgga 5880 aaagcctaac ctttaccagg tccgcctgta tttataccag gcaggcagcc tcttagaggt 5940 tagccggcag gaagtgggtt tccgcaactt tgaactaaaa gacgggatta tgtaccttaa 6000 cggccagcgg atcgtcttca agggggccaa ccggcacgaa tttgacagta agttgggtcg 6060 ggctatcacg gaagaggata tgatctggga catcaagacc atgaagcgaa gcaacatcaa 6120 tgctgtccgc tgctctcact acccgaacca gtccctcttt taccggctct gtgacaagta 6180 cggcctttac gtcattgatg aagctaacct ggaaagccac ggcacctggg aaaaagtggg 6240 ggggcacgaa gatcctagct tcaatgttcc aggcgatgac cagcattggc tgggagccag 6300 cttatcccgg gtgaagaaca tgatggctcg ggacaagaac catgcttcaa tcctaatctg 6360 gtctttaggc aatgagtctt acgccggcac tgtctttgcc caaatggctg attacgtccg 6420 gaaggctgat ccgacccggg ttcagcacta tgaaggggtg acccacaacc ggaagtttga 6480 cgacgccacc cagattgaaa gccggatgta tgctccggcc aaggtaattg aagaatactt 6540 gaccaataaa ccagccaagc catttatctc agttgaatac gctcacgcca tgggcaactc 6600 cgtcggtgac ctggccgcct acacggccct ggaaaaatac ccccactacc agggcggctt 6660 catctgggac tggattgacc aaggactgga aaaagacggg cacctgcttt atgggggcga 6720 cttcgatgac cggccaaccg actatgaatt ctgcgggaac ggcctggtct ttgctgaccg 6780 gactgaatcg ccgaaactgg ctaatgtcaa ggccctttac gccaacctta agttagaagt 6840 aaaagatggg cagctcttcc tcaaaaacga caatttattt accaacagct catcttacta 6900 cttcttgact agtcttttgg tcgatggcaa gttgacctac cagagccggc ctctgacctt 6960 tggcctggag cctggcgaat ccgggacctt tgccctgcct tggccggaag tcgctgatga 7020 aaaaggggag gtcgtctacc gggtaacggc ccacttaaaa gaagacttgc cttgggcgga 7080 tgagggcttc actgtggctg aagcagaaga agtagctcaa aagctgccgg aatttaagcc 7140 ggaagggcgg ccagatttag ttgattccga ctacaaccta ggcctgaaag gaaataactt 7200 ccaaattctc ttctccaagg tcaagggctg gccggtttcc ctcaagtatg ccggtaggga 7260 atacttgaag cggctgccgg aatttacctt ctggcgggcc ctgacggaca acgaccgggg 7320 agctggttac ggctatgatc tggcccggtg ggaaaatgcc ggcaagtatg cccgcttgaa 7380 agacatcagc tgcgaggtca aggaagactc cgttttggtc aagactgcct ttacgttgcc 7440 tgtcgcctta aagggtgatt taaccgtgac ctatgaagtc gatggacggg gcaagattgc 7500 tgtaacagct gacttcccag gcgcggaaga agctggtctc ttgccagcct ttggcttgaa 7560 cctggccctg ccaaaagaac tgaccgatta ccgctactat ggtctgggac ctaatgagag 7620 ctacccagac cgcttggaag gtaattacct gggcatctac cagggagcgg taaaaaagaa 7680 ctttagccca tatcgtccgc aggaaacggg caaccggagc aaggttcgct ggtaccagct 7740 ctttgatgaa aagggcggct tggaatttac ggccaatggg gcagacttga acttgtctgc 7800 tttgccatat tctgccgccc aaattgaagc agcggaccac gcttttgaac tgactaacaa 7860 ttacacttgg gttagagcct taagcgccca gatgggggtc ggcggggatg actcctgggg 7920 gcagaaggtc cacccggaat tctgcctgga tgctcaaaaa gcccgccagc ttcgcctggt 7980 gattcagccc cttttactaa aataaatgct acaattgact taacaggatg aaattttagt 8040 aaaagcaaag cgagtgagga agatggcaac gatcagagaa gtgccaaggc agccggcgtg 8100 tcgctagcga cggtc 8115 2 8115 DNA Lactobacillus plantarum 2 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca 60 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta 120 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat 180 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga ctatttacgg 240 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac 300 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt 360 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg 420 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc 480 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt 540 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 600 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg 660 actcactata gggagaccca agcttggtac cgagctcgga tccactagta acggccgcca 720 gtgtgctgga attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag 780 ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct 840 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 900 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 960 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac 1020 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc 1080 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 1140 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 1200 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 1260 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 1320 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 1380 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 1440 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 1500 cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 1560 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 1620 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 1680 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 1740 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 1800 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 1860 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 1920 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 1980 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2040 cccgaaaagt gccacctgac gtcgacggat cgggagatca tatcctgaca ttctctttac 2100 caaataaaat aattttgttt attaaaatcc cattttgcga caacttcttc cgcagcttcc 2160 atttgctctt tggtgtaatc ttcatcgcca acatgaacta aatcaccatt ctcaacatct 2220 tcaagtttca aatcttgctt aatttgcttt aataatccac catagctgat ttgtcgtgtt 2280 ccagctaagg catactccaa atttttaatc accaccaaat tacgctcatc atcagccgtc 2340 atataatcag ctgattttac ctcgtatttc gccgtttctt cggcactagc ttgcaaagag 2400 tcagttcctt tacgtttgtt agctttaaca gcctgcacat gcaccacagg ctcataatca 2460 actttcaagg ctttttgcca taattttgcc cattctgctt gtgctaaata attatttgaa 2520 ttcttaaaat aacttgattt tacaaacagc aacacatgca agtgttgatt atatgacccg 2580 tcttgttcat taacggtaat ttccgttgaa cgtaaataac ccaataaatt tttagtcact 2640 tttttatagc gagttagctt attaaaggct ttagtcaaag ctcttaaaga cacttttaac 2700 tcctctgctg aatgagcgtt tttaacggtt aaagttaaaa acaaaaaccg tcctttaggc 2760 tctcttgcaa ctgcttccgc aataatttgt tttaactggc tcgagttttt catgctcctt 2820 ctccaattac acaatggaca caatcgttta tgacaaaacc acgtttgata aagttttaag 2880 tgctcgccaa tcttacgaaa acgcaaaact tcaccacaac cccgtacatc atgtgcccgt 2940 ttaaattcta agattgccaa atattcggca tagcgcacat tttcaatctt ccgttctcgc 3000 caaggtctaa ctttgccatt ttcagtttta tcttcaaaaa tttctgacat aaaaagctcc 3060 tccagtttat ccacgtgaag gagctgacta tctttttcaa taagcttata accttgacat 3120 catagggctt ttcccctaga ataggctata aatcgcaaat gataatcaac tcacgtgttc 3180 cgagcggcca aactaggaat ttgcacgtgg gtttttattt tgtctttctt tcaaccaatt 3240 tataacccta ataatacacc aaaagcctat aaaatcaatg gatacaagcc caattaagcc 3300 taatcaagct tgattttaaa aaactagttg ttgctaatag tatcaagata agaagaaaac 3360 gccaaaaatt gcgtttttaa accccaaaaa gcagatcagc aaaaaccgct gaactgcttt 3420 ttttaaaccg tggctttcag ccacactgac cagctgaacc agctggaccg taacgcttgc 3480 cgccgctggg ctcgggaaaa caagggcttg ttttccaaga cgtcaggctt ttggtattgt 3540 ctagtctatc aactccttaa agcctccaag aggggctaat atcgcctgta aggctcaata 3600 agcccctcta agtcgattta ccgttgacag acagttagat agctaactgt tagctaaaat 3660 cgcttagaac gcaaataaga gcctttaaaa ttaacgttca aaaataaaaa agttcgaagg 3720 agctagcgac tgaacttatt tatttttgaa tgttccaaac tgacgcaagt cagttacgtt 3780 tgagcaacgc gaaatctgat gcaggttttg atgggtttag cacaacacaa cttcatgttg 3840 tgtgtaagtg cgcactacat gataatgcgc actacatgat aatgcgcact acatgataat 3900 gtgcgcacta catgataatg cgcactacat gataatgtac atgataatgt gcgcactaca 3960 tgataatgcg cactacatga taatgcgcac tacatgataa tgcgcactac atgataatgc 4020 gcactacatg ataatgcgca ctacatgata atgcgcacta catgataatg tgcacttaca 4080 ctccaaataa attggagtaa tgctaaaacc tgtatcagaa gtcagcaagc tgacaacaaa 4140 aaagggatat gccaacggat ttaccgttga tctcccgatc ccctatggtc gactctcagt 4200 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag 4260 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat 4320 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgttagaa gcaaactaag 4380 agtgtgttga gtagtgcagt atcttaaaat tttgtataat aggaattgaa gttaaattag 4440 atgctaaaaa tttgtaatta agaaggagtg attacatgat tggcagccag tctccgggca 4500 attaatgaac ttggacatgg ttgacgaccc ggtctttgca agccgaattc gaccacactg 4560 gcggccgtta ctagggtatc gatccgataa aaagttaggc gacggctttg ccctggtgcc 4620 agcagacggt aaggtctacg cgccatttgc cggtactgtc cgccagctgg ccaagacccg 4680 gcactcgatc gtcctggaaa atgaacatgg ggtcttggtc ttgattcacc ttggcctggg 4740 cacggtcaaa ttaaacggga ctggctttgt cagctatgtt gaagagggca gccaggtaga 4800 agccggccag cagatcctgg aattctggga cccggcgatc aagcaggcca agctggacga 4860 cacggtaatc gtgaccgtca tcaacagcga aactttcaca aatagccaga tgctcttgcc 4920 gatcggccac agcgtccaag ccctggatga tgtattcaag ttagaaggga agaattagaa 4980 aatgagcaat aagttagtaa aagaaaaaag agttgaccag gcagacctgg cctggctgac 5040 tgacccggaa gtttacgaag tcaatacaat tcccccgcac tccgaccatg agtccttcca 5100 aagccaggaa gaactggagg agggcaagtc cagtttagtg cagtccctgg acggggactg 5160 gctgattgac tacgctgaaa acggccaggg accagtcaac ttctatgcag aagactttga 5220 cgatagcaat tttaagtcag tcaaagtacc cggcaacctg gaactgcaag gctttggcca 5280 gccccagtat gtcaacgtcc aatatccatg ggacggcagt gaggagattt tcccgcccca 5340 aattccaagc aaaaatccgc tcgcttctta tgtcagatac tttgacctgg atgaagcttt 5400 ctgggacaag gaagtcagct tgaagtttga cggggcggca acagccatct atgtctggct 5460 gaacggccac ttcgtcggct acggggaaga ctcctttacc ccaagcgagt ttatggttac 5520 caagttcctc aagaaagaaa ataaccgcct ggcagtggct ctctacaagt attcttccgc 5580 ctcctggctg gaagaccagg acttctggcg catgtctggt ttgttcagat cagtgactct 5640 tcaggccaag ccgcgtctgc acttggagga ccttaagctt acggccagct tgaccgataa 5700 ctaccaaaaa ggaaagctgg aagtcgaagc caatattgcc taccgcttgc caaatgccag 5760 ctttaagctg gaagtgcggg atagtgaagg tgacttggtt gctgaaaagc tgggcccaat 5820 cagaagcgag cagctggaat tcactctggc tgatttgcca gtagctgcct ggagcgcgga 5880 aaagcctaac ctttaccagg tccgcctgta tttataccag gcaggcagcc tcttagaggt 5940 tagccggcag gaagtgggtt tccgcaactt tgaactaaaa gacgggatta tgtaccttaa 6000 cggccagcgg atcgtcttca agggggccaa ccggcacgaa tttgacagta agttgggtcg 6060 ggctatcacg gaagaggata tgatctggga catcaagacc atgaagcgaa gcaacatcaa 6120 tgctgtccgc tgctctcact acccgaacca gtccctcttt taccggctct gtgacaagta 6180 cggcctttac gtcattgatg aagctaacct ggaaagccac ggcacctggg aaaaagtggg 6240 ggggcacgaa gatcctagct tcaatgttcc aggcgatgac cagcattggc tgggagccag 6300 cttatcccgg gtgaagaaca tgatggctcg ggacaagaac catgcttcaa tcctaatctg 6360 gtctttaggc aatgagtctt acgccggcac tgtctttgcc caaatggctg attacgtccg 6420 gaaggctgat ccgacccggg ttcagcacta tgaaggggtg acccacaacc ggaagtttga 6480 cgacgccacc cagattgaaa gccggatgta tgctccggcc aaggtaattg aagaatactt 6540 gaccaataaa ccagccaagc catttatctc agttgaatac gctcacgcca tgggcaactc 6600 cgtcggtgac ctggccgcct acacggccct ggaaaaatac ccccactacc agggcggctt 6660 catctgggac tggattgacc aaggactgga aaaagacggg cacctgcttt atgggggcga 6720 cttcgatgac cggccaaccg actatgaatt ctgcgggaac ggcctggtct ttgctgaccg 6780 gactgaatcg ccgaaactgg ctaatgtcaa ggccctttac gccaacctta agttagaagt 6840 aaaagatggg cagctcttcc tcaaaaacga caatttattt accaacagct catcttacta 6900 cttcttgact agtcttttgg tcgatggcaa gttgacctac cagagccggc ctctgacctt 6960 tggcctggag cctggcgaat ccgggacctt tgccctgcct tggccggaag tcgctgatga 7020 aaaaggggag gtcgtctacc gggtaacggc ccacttaaaa gaagacttgc cttgggcgga 7080 tgagggcttc actgtggctg aagcagaaga agtagctcaa aagctgccgg aatttaagcc 7140 ggaagggcgg ccagatttag ttgattccga ctacaaccta ggcctgaaag gaaataactt 7200 ccaaattctc ttctccaagg tcaagggctg gccggtttcc ctcaagtatg ccggtaggga 7260 atacttgaag cggctgccgg aatttacctt ctggcgggcc ctgacggaca acgaccgggg 7320 agctggttac ggctatgatc tggcccggtg ggaaaatgcc ggcaagtatg cccgcttgaa 7380 agacatcagc tgcgaggtca aggaagactc cgttttggtc aagactgcct ttacgttgcc 7440 tgtcgcctta aagggtgatt taaccgtgac ctatgaagtc gatggacggg gcaagattgc 7500 tgtaacagct gacttcccag gcgcggaaga agctggtctc ttgccagcct ttggcttgaa 7560 cctggccctg ccaaaagaac tgaccgatta ccgctactat ggtctgggac ctaatgagag 7620 ctacccagac cgcttggaag gtaattacct gggcatctac cagggagcgg taaaaaagaa 7680 ctttagccca tatcgtccgc aggaaacggg caaccggagc aaggttcgct ggtaccagct 7740 ctttgatgaa aagggcggct tggaatttac ggccaatggg gcagacttga acttgtctgc 7800 tttgccatat tctgccgccc aaattgaagc agcggaccac gcttttgaac tgactaacaa 7860 ttacacttgg gttagagcct taagcgccca gatgggggtc ggcggggatg actcctgggg 7920 gcagaaggtc cacccggaat tctgcctgga tgctcaaaaa gcccgccagc ttcgcctggt 7980 gattcagccc cttttactaa aataaatgct acaattgact taacaggatg aaattttagt 8040 aaaagcaaag cgagtgagga agatggcaac gatcagagaa gtgccaaggc agccggcgtg 8100 tcgctagcga cggtc 8115 3 30 DNA Artificial Sequence oligonucleotide for PCR 3 aagctcatga ttggcagcca gtctccgggc 30 4 30 DNA Artificial Sequence oligonucleotide for PCR 4 gacctcatga accgtcgcta gcgacacgcc 30 5 31 DNA Artificial Sequence oligonucleotide for PCR 5 ttaacgatcg ttagaagcaa acttaagagt g 31 6 27 DNA Artificial Sequence oligonucleotide for PCR 6 ttaacgatcg atgtaatcac tccttct 27 

What is claimed is:
 1. A Lac shuttle vector, comprising: (a) a region which regulates a plasmid copy number, wherein said region comprises an E. coli replication origin sequence; (b) an eukaryotic gene expression cassette, which comprises an eukaryotic gene transcriptional promoter sequence, a multiple cloning site and a transcriptional terminator sequence, wherein a heterologous gene is inserted into said multiple cloning site; (c) a lactic acid bacteria plasmid sequence, which comprises a plus origin of replication, and a nucleic acid sequence encoding for a protein which relates to the lactic acid bacteria plasmid replication; and (d) a non-antibiotic resistance selection gene and the promoter sequence thereof.
 2. The Lac shuttle vector as claimed in claim 1, wherein said eukaryotic gene transcriptional promoter is cytomegalovirus (CMV) promoter.
 3. The Lac shuttle vector as claimed in claim 1, wherein said lactic acid bacteria plasmid sequence is the plasmid of 2.1 kb size isolated from Lactobacillus plantarum.
 4. The Lac shuttle vector as claimed in claim 3, wherein the protein which relates to the lactic acid bacteria plasmid replication is Rep A protein containing 317 amino acids.
 5. The Lac shuttle vector as claimed in claim 1, wherein said non-antibiotic resistance selection gene is β-galactosidase gene.
 6. The Lac shuttle vector as claimed in claim 5, wherein the promoter of said β-galactosidase gene is erythromycin resistance gene promoter.
 7. The Lac shuttle vector as claimed in claim 1, wherein the Lac Shuttle vector comprises the nucleotide sequence set forth in SEQ ID NO:1 or a complementary nucleotide sequence thereto, or a degenerate variant thereof.
 8. The Lac shuttle vector as claimed in claim 1, wherein the Lac Shuttle vector comprises the nucleotide sequence set forth in SEQ ID NO:2 or a complementary nucleotide sequence thereto, or a degenerate variant thereof.
 9. The Lac shuttle vector as claimed in claim 1, wherein the Lac Shuttle vector is selected from the group consisting of: (a) pCLP7 (having th e configuration of restriction sites in FIG. 3, ATCC Accession No. PTA-2661); and (b) PCLP8 (having the configuration of restriction sites in FIG. 3, ATCC Accession No. PTA-2663).
 10. The Lac shuttle vector as claimed in claim 1, wherein the host cell being transformed is a Gram-positive bacterium, and the endogenous β-galactosidase gene of the host cell is not capable of producing a normal enzymatic function.
 11. The Lac shuttle vector as claimed in anyone of claim 10, wherein the host cell is the mutant of Lactobacillus casei (subsp. casei), which is designated Ana-1 (Lac⁻ mutant), (ATCC Accession No. PTA-2662).
 12. A kit for expression of a heterologous gene, comprising: (a) the Lac shuttle vector as claimed in claim 1; (b) a host cell which the endogenous β-galactosidase gene thereof is not capable of producing a normal enzymatic function; and (c) an eukaryotic cell.
 13. A DNA vaccine carrier comprising the Lac shuttle vector as claimed in claim
 1. 14. A method for selection of a host cell containing a vector, comprising: (i) introducing into said host cell the Lac shuttle vector as claimed in claim 1, wherein the endogenous β-galactosidase gene of said host cell is not capable of producing a normal enzymatic function; and (ii) culturing said host cell transformed in step (i) under conditions which lactose is the only carbon source. 